Paramecium jenningsi Species Complex (Ciliophora, Protista) in India:
the First Description of New Stands of P. bijenningsi and P. trijenningsi
Ewa PRZYBOŒ, Chaganti KALAVATI, Sergei I. FOKIN, and Sebastian TARCZ
Accepted February 01, 2017 Published online April 24, 2017 Published April 28, 2017
PRZYBOŒE., KALAVATI C., FOKINS.I., TARCZsincerely. 2017. Paramecium jenningsi
species complex (Ciliophora, Protista) in India: the first description of new stands of
P. bijenningsi and P. trijenningsi. Folia Biologica (Kraków) 65: 55-61.
Paramecium jennngsi (Ciliophora, Protista) is a complex of three cryptic species known from
13 sampling points situated around the world, mainly in the tropics. Two strains recently collected in India were identified as P. bijenningsi and P. trijenningsi from the P. jenningsi complex, based on an analysis of 16 (both nuclear and mitochondrial) loci, strain crosses, and cytological analyses. Current results increase the knowledge about the species range of particular members of the P. jenningsi complex.
Key words: Ciliates, Paramecium jenningsi species complex, multi-loci analysis, strain crosses, cytological analysis.
Ewa PRZYBOŒ, Sebastian TARCZ, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków 31-016, S³awkowska 17, Poland.
E-mail: przybos@isez.pan.krakow.pl
Chaganti KALAVATI, Department of Zoology, Andhra University, Visakhapatnam, India. Sergei I. FOKIN, Department of Biology, University of Pisa, Pisa, Italy.
Microbial eukaryotes are one the of the main components of tropical food webs in different eco-systems. However, our knowledge of their biodiver-sity is still significantly underestimated (M ED-INGERet al. 2010). Even in Paramecium (one of
the best studied genera of microbial eukaryotes), the problem of under-sampling is still a concern in less frequently studied parts of the world, particu-larly the southern hemisphere (FOKIN2010/2011). Similarly, data concerning the occurrence of par-ticular species from the tropics are rare. One of the
Paramecium morphospecies with preference for
a warm climate and therefore poorly studied as con-cerns its range and biodiversity is P. jenningsi (DILLER & EARL1958). Although up to now it was collected in only 13 sampling sites distributed worldwide but situated mainly in the tropics (PRZYBOŒet al. 1999), it was suggested (PRZYBOŒ
et al. 1999, 2003; MACIEJEWSKA2007) and then confirmed by strain crosses, morphological and molecular analyses (PRZYBOŒ& TARCZ2013) to be a complex of three cryptic species (PRZYBOŒ& TARCZ2016). Moreover, two of them are known
from one (P. primjenningsi) or two (P. bijenningsi) southern Asian locations in contrast to P. trijenningsi collected from the Japanese islands (6 sampling points), Africa (2 sampling points) and Americas (2 sampling points). With reference to the above, every new sampling point of P. jenningsi complex should bring key information about its biogeogra-phy and cryptic diversity. In the present study, based on strain crosses, cytological analyses as well as a comparison of 11 nuclear and 5 mitochondrial loci, we showed the Paramecium species affilia-tion of two strains collected in India in 2014.
Material and Methods Material
The two investigated strains (designated KT1 and MVP1) of the P. jenningsi complex (Table 1) originated from India, Andhra Pradesh, Visak-hapatnam. More precisely, the KT1 strain was col-lected in the Kottura, a freshwater pond (18° 5¢34.76"N, Ó Institute of Systematics and Evolution of Animals, PAS, Kraków, 2017
Open Access article distributed under the terms of the Creative Commons Attribution License (CC-BY)
83° 8¢10.56"E) on 27/01/2014. The MVP1 strain was sampled in a wastewater stream in MVP Col-ony, which is an urban neighbourhood of the city of Visakhapatnam (17°44¢14.63"N, 83°20¢16.79"E) on 24/03/2014. The general morphology and nu-clear apparatuses in vegetative and autogamous individuals are characterized by cytological prepa-rations (presented in Fig. 1), and the dimensions of particular strains are given in Table 3.
Strain cultivation
Paramecia were cultured in a medium made of dried lettuce and distilled water inoculated with
Enterobacter aerogenes according to the methods
of SONNEBORN (1950, 1970) and supplemented with 0.8 µg/ml ß sitosterol (Merck, Darmstadt, Germany). Methods used in cytological studies, strain measurements, and crosses, as well as molecular analyses were as in PRZYBOŒand TARCZ(2016).
Molecular methods and data analysis
Paramecium genomic DNA was isolated from
vegetative cells at the end of the exponential phase (approx. 1000 cells were used for DNA extrac-tion). Eleven nuclear and five mitochondrial DNA fragments were amplified, sequenced and ana-lyzed. The above techniques as well as the nucleo-tide sequences of the primers used for amplification and sequencing were described in detail in PRZYBOŒand TARCZ(2016). The DNA sequences of both newly identified strains from In-dia are available from the NCBI GenBank data-base (see Table 2). The studied sequences were aligned using ClustalW (THOMPSONet al. 1994)
as part of BioEdit software (HALL 1999) and checked manually. All of the obtained sequences were unambiguous and incorporated in analysis. Phylograms were constructed for the studied frag-ments as in PRZYBOŒand TARCZ(2016).
Table 1 Strains of the Paramecium jenningsi complex used in present studies
No Species Strainindex Strain geographicalorigin Collector’s name Reference
1. Paramecium bijenningsi MVPI India, Andra Pradesh Valentina SERRA
and Charan Kumar BASURI unpublished
2. Paramecium trijenningsi KTI India, Andra Pradesh Sergei I. FOKIN, Valentina SERRA
and Charan Kumar BASURI SERRAet al. 2014
Fig. 1. A – strain MVPI of P. bijenningsi. Vegetative individual, macronucleus (Ma), two micronuclei (mi). B – strain MVPI of P. bijenningsi. Autogamous individual, fragmented old macronucleus (OMa), two macronuclear anlagen (A) with chromatin centre. C – strain KTI of P. trijenningsi. Vegetative individual, macronucleus (Ma), two micronuclei (mi). D – strain KTI of P. trijenningsi. Autogamous individual, fragmented old macronucleus (OMa), two macronuclear anlagen (A) with chromatin centre.
Results and Discussion
Cytological characteristics of strains from the
P. jenningsi complex
The strains are characterized by two micronuclei (Figs 1A,C) of chromosomal type with a reticular structure (FOKIN1997; PRZYBOŒet al. 2015) and
two macronuclear anlagen with chromatin centres (DILLER& EARL1958; MITCHELL1963; PRZYBOŒ
1975, 1986; PRZYBOŒet al. 2003; PRZYBOŒ& TARCZ
2013) seen at a later stage of nuclear reorganiza-tion in sexual processes (autogamy, conjugareorganiza-tion) (Figs 1B,D). Paramecia have the shape of a cigar, with two contractive vacuoles on the dorsal side, usually with 8 long radial canals.
Dimensions of strains
The dimensions (length and width of cells and macronuclei) of the strains (fixed, Giemsa-stained) and diameter of micronuclei are given in Table 3. The average length of the cells in the strain MVPI equals 248.03 ìm, and 228.15 ìm in the strain KTI. The diameter of micronuclei did not show much variation between the strains (3.12 - 3.85ìm in both strains).
Biology – the appearance of sexual processes Autogamy (leading to homozygosity of all genes) was observed in strains that were starved for 16-20 fissions (as an inter-autogamous period between successive autogamies) in daily isolated lines (d.i.l.) cultivated at 27°C in a medium ena-bling three fissions daily. In turn, conjugation was obtained on the sixth or seventh day of clone cul-ture at 27°C at a rate of three fissions daily. The cy-toplasmic type of mating type inheritance is characteristic for the species.
Identification of species and results of strain crosses
Strain MVPI was identified as P. bijenningsi and strain KTI as P. trijenningsi, based on conjugation between the studied strains and the reference strains of the particular species, i.e. the strain CS from China, Shanghai, reference for P. bijenningsi, and the strain JH from Japan, Honshu Island, Hagi, reference for P. trijenningsi. The percentage of surviving hybrid clones in F1 and F2 was high (Ta-ble 4). The results of the strain crosses are in con-cordance with the results of multi-locus analyses of the studied strains of the P. jenningsi complex (Figs 2,3). Table 2 GenBank accession numbers to studied D NA fragments nucDNA 1 nucDNA 2 nucDNA 3 nucDNA 4 nucDNA 5 nucDNA 6 nucDNA 7 nucDNA 8 nucDNA 9 nucDNA 10 nucDNA 11 mtDNA 1 mtDNA 2 mtDNA 3 mtDNA 4 mtDNA 5 rDNA Msh2 tRNAmet Ubiq PTMB.21c PTMB.42 PTMB.159 PTMB.376 PTMB.377 PTMB.412 PTMB.418 COI CytB COII 3’COI-inter genic region rns a 1. Paramecium bijenningsi , strain MVP1 KY635365 KY635367 KY635369 KY635371 KY635373 KY635375 KY635377 KY635379 KY635381 KY635383 KY635385 KY635387 KY635389 KY635391 KY635393 KY63539 5 2. Paramecium trijenningsi , strain KTI KY635364 KY635366 KY635368 KY635370 KY635372 KY635374 KY635376 KY635378 KY635380 KY635382 KY635384 KY635386 KY635388 KY635390 KY635392 KY63539 4
Table 3 Dimensions of studied strains of the P. jenningsi complex
No Strain,species Average length of cells (µm) SD Average cell width (µm) SD Average length of MAC (µm) SD Average width of MAC (µm) SD Average diameter of MIC (µm) SD 1. P. bijenningsi, MVPI 248.03 26.14 88.22 9.61 56.40 7.17 23.64 4.46 3.12 0.41 2. P. trijenningsi,KTI 228.15 22.78 80.66 11.32 58.46 8.76 17.40 3.85 3.01 0.53 Table 4 Mean percentage of surviving clones (F1/F2generations) in strain crosses within the species
of the Paramecium jenningsi complex
Species Strain designation F1 obtained by crosses F2 obtained by autogamy
P. bijenningsi MVPI (India) x CS (China) 91 82
P. trijenningsi KTI (India) x JH (Japan) 98 94
Fig. 2. Unrooted phylogram for 15 P. jenningsi complex strains and 16 other strains of the subgenus Paramecium constructed on the basis of a comparison of eleven concatenated nuclear genome fragments using the neighbor joining method. Bootstrap values for neighbor joining, maximum parsimony, and maximum likelihood analysis, as well as posterior probabilities for Bayesian inference are presented. Bootstrap values of less than 50% (posterior probabilities of less than 0.50) are not shown. All positions containing gaps and missing data were eliminated. There were a total of 3922 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 6.0 (NJ/MP/ML) and MrBayes 3.1.2 (BI).
Phylogenetic analysis
All applied methods (NJ – neighbour-joining, MP – maximum parsimony, ML – maximum like-lihood, BI – Bayesian inference) of tree recon-struction gave an almost identical topology, so in the current study only neighbour-joining phy-lograms (Figs 2, 3) providing bootstrap/posterior probability values for the other methods are pre-sented. Due to the lack of nuclear sequences for
P. caudatum (outgroup) it was impossible to
ob-tain a rooted tree for the nucDNA dataset. Based on the two phylogenetic trees (Figs 2, 3) constructed on the basis of two datasets (nucDNA and mtDNA) of 16 sequenced genome fragments, the division of P. jenningsi into two clades (P.
bijenningsi and P. trijenningsi ) and one branch (P. primjenningsi) can be seen. The strains from India
identified by cytological characteristics as P.
jen-ningsi appear with strong bootstrap support in both
trees in two different clades: strain MVPI in the P.
bijenningsi cluster and strain KTI in the P.
trijen-ningsi cluster. The positions of MVPI and KTI
strains (Figs 2,3) are in accordance with the results of strain crosses (Table 4). It is worth noting that strain MVPI based on a comparison of the nucDNA dataset (Fig. 2) is most closely related to the P. bijenningsi strain from Saudi Arabia (SA) but in the case of the mtDNA dataset (Fig. 3) it ap-pears in one subclade with the P. bijenningsi strain from China (CS). A similar situation was observed in the case of the second Indian strain (KTI) which appears in one subclade with the Uganda strain (U) (Fig. 2 – nucDNA tree) and with the Panama strain (PA) (Fig. 3 – mtDNA tree). However, the ob-served positions did not influence species identifi-cation of either of the strains.
Species identification of new Paramecium strains Due to the absence of a uniform species defini-tion, the abundance of cryptic diversity, and the occurrence of convergent morphology (BOENIGK Fig. 3. Phylogram or 15 P. jenningsi complex strains and 17 other strains of the subgenus Paramecium (the CA strain of P. caudatum was used as an outgroup) constructed on the basis of a comparison of five concatenated mitochondrial genome fragments using the neighbor joining method. Bootstrap values for neighbor joining, maximum parsimony, and maximum likelihood analysis, as well as posterior probabilities for Bayesian inference are presented. Bootstrap values of less than 50% (posterior probabilities of less than 0.50) are not shown. All positions containing gaps and missing data were eliminated. There were a total of 2553 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 6.0 (NJ/MP/ML) and MrBayes 3.1.2 (BI).
et al. 2012), different approaches to demarcating
boundaries between protist species should be ap-plied (CARON2013). Likewise in the current sur-vey, except existing cryptic differentiation in P.
jenningsi morphospecies, the occurrence of
para-phyly between P. aurelia and P. jenningsi com-plexes (PRZYBOŒet al. 2015; PRZYBOŒ& TARCZ
2016; this study Figs 2, 3) forced application of morphological studies, strain crosses and multi-locus analysis for proper species identification. Based on the obtained results both strains from Andra Pradesh, India are identified as P. bijen -ningsi (strain MVPI) and P. trijen-ningsi (strain
KTI) – members of the P. jenningsi species com-plex (cf PRZYBOŒ& TARCZ2016). What is more, the first description of P. jenningsi (DILLER & EARL1958) was also based on a strain from Ban-galore, India. At present, it is recognized as P.
primjenningsi (PRZYBOŒ& TARCZ2016) of the P.
jenningsi complex. The studied herein Parame-cium species meet the criteria of a species complex
(according to BICKFORDet al. 2007) because they
can be differentiated based on strain crosses and molecular characteristics but they cannot be dif-ferentiated based on morphological features alone. The strains identified in this work as P. bijenningsi and P. trijenningsi were collected and described as
P. jenningsi (SERRAet al. 2014) but without
de-tailed identification of cryptic species. In 2014 an-other Indian strain (designated BJ1) was collected in Chilka Lake, Odisha, presenting a macronuclear endosymbiont “Candidatus Gortzia infectiva” (SERRAet al. 2016), a recently described bacterial
species originally retrieved in P. jenningsi strain TS-j1-8 from Thailand (BOSCARO et al. 2013).
Based on the results presented here, the future sampling of tropical regions such as India, which are in general poorly investigated in terms of
Para-mecium diversity, may reveal new data on
biodi-versity and biogeography of known or even undiscovered morpho-species.
Acknowledgements
International mobility of C. KALAVATI, S.I. FOKIN, V. SERRAand C. K. BASURIwas supported by the European Commission FP7-PEOPLE-2009-IRSES project CINAR PATHOBACTER (247658), co-ordinator G. PETRONI. The authors are grateful to the Marine Biology Laboratory, Andhra Univer-sity, India, for providing the research facilities. In particular, Prof. Raman AKKUR, Prof. Bhagava-tula V. SANDEEPand Prof. Prabhakara Rao Y AL-LAPRAGADAare acknowledged for their valuable support. Special acknowledgments to Valentina SERRA and Charan Kumar BASURI who carried out the sampling in India.
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